Experimental Study Of Ipsilateral C7 Nerve Root Transfer To Repair The Root Avulsion Of C5 And C6 Of The Brachial Plexus

Part 1 An experimental study on treatment outcome foripsilateral C7 nerve root transfer to repair the root avulsion of C5and C6 of the brachial plexusObjective Multiple extraplexal nerve transfers were frequently carried out totreat root avulsion of C5 and C6 of the brachial plexus.Gu YD(2002) first used anintraplexal transfer,that is the ipsilateral C7 root transferred to the upper trunk,totreat that kind of lesion.The following study was to experimentally compare thetreatment outcome of the injured upper limb for the C5-C6 root avulsion of thebrachial plexus repaired by ipsilateral C7 nerve root transfer and other threecommonly used nerve transfers.Methods 120 SD rats were randomly divided into four groups 30 each.All ofthem were fastened supine and anesthetized,then a midline cervical incision wasmade.After pectoralis major was incised along the arcuate line,pectoralis minor wasretracted laterally,and C5-T1 nerve roots were exposed between anterior and middlescalenus muscles.After C5 and C6 root was avulsed,each group underwent variouscombined nerve transfers,that is,(1)'Ipsilateral C7'group:the ipsilateral C7 roottransferred to the upper trunk of brachial plexus and the spinal accessory nerve to thesuprascapular nerve.(2)'Oberlin'group:partial fascicles of the ulnar nerve transferredto the biceps branch(Oberlin's procedure),the spinal accessory to the suprascapularand branches to the triceps long head to the axillary nerve.(3)'phrenic nerve'group:the phrenic transferred to the musculocutaneous,cervical plexus motor branches tothe lower trunk(axillary nerve) of brachial plexus and the spinal accessory nerve to thesuprascapular nerve.(4)'forsake repairing axillary nerve'group:the phrenictransferred to the musculocutaneous and the spinal accessory nerve to thesuprascapular nerve.Neurotization outcomes were evaluated at 3,6 and 12 weeks postoperatively by comparing changes of behavioral tests,neurophysiologicalinvestigations,muscular histology,and muscular functional examinations.Theparameters were listed as follows:(1) Ochiiai clinical scores:The clinical score wascalculated under two condition.First,the rats were held by the tail above a table,andthen they were placed on table to compare the printed walking patterns.(2) Barthfoot-fault test:the rat was placed on a horizontal grid floor.The foot-fault wasdetermined by an animal misplacing a forelimb and a paw falling through the gridbars.The number of forelimb faults occurring in 2 min was counted.(3) Terzisgrooming test:Squirting 1 to 3 ml of water over the rat's snout,which always elicitedbilateral grooming movements from the rats.We were able to instantaneously assessfunctioning of the experimental side and compare it with the normal side.(4)Compound muscle action potential(CMAP):Maximal current and single square-wavewere used to stimulate the musculocutaneous nerve and axillary nerve.The needleelectrodes were inserted 2 mm-depth for the biceps and deltoid muscles,thestimulating and recording electrodes were placed in the same position at bothforelimbs of rats,and the CAMP amplitude and latency were recorded.(5) Musclefunction,wet weight and cross-sectional area of muscle fiber.The distal portions ofbiceps and deltoid muscles were free,transected and attached to the force transducter.The musculocutaneous and axillary nerves were simulated respectively with asuper-high voltage and a high frequency series of square wave impulses.The curvesof maximum titanic contractile tension of isometric contraction were recorded on atwo-conduction physiological recorder.And then,the biceps and deltoid muscles wereharvested completely by removing connective tissues,then weighed by AnalyticBalance.Transverse sections of those muscle samples were stained by hematoxylinand eosin (HE),and image analysis system was used to measure the cross-sectionalarea of muscle fibers.(6) Regenerating myelinated axon counts.Musculocutaneousnerve and axillary nerve specimens were taken around the anastomotic site.Sectionsof each nerve were stained with toluidine blue and processed for computer-aidedimage analysis system of regenerative axon number.The left forelimbs of both grouprats were taken as the experimental sides,and the right forelimbs of them were takenas the control sides.Values of the control side were taken as 1,so that the recoveryrates of the left side could be obtained by the parameters of the left forelimb dividedby those of the fight forelimb.Wilcoxon signed-ranks test was used for the ratesbetween the'ipsilateral C7'group and the other 3 control groups respectively. Bonferroni adjustment was used for multiple comparisons.All of the reported Pvalues were two-tailed,and values of p＜0.017 were considered significant.Results 3 weeks after operation,no significant difference were found betweenthe'ipsilateral C7'group and other three control groups in the three behavioralevaluations (P＞0.017).Neurophysiologic investigations of the axillary nerve showedthe'ipsilateral C7'group was superior to the other three groups (Z-value were 3.184,3.593,3.672,3.104,3.266,3.674 respectively,P＜0.017) .Muscular histological andfunctional outcome of the axillary nerve and deltoid muscle showed the'ipsilateralC7'group was superior to the'phrenic nerve'(Z-value were 3.628,3.103,3.115,3.943respectively,P＜0.017) and'forsake repairing axillary nerve'groups (Z-value were3.674,3.593,3.429,3.296 respectively,P＜0.017) ,while no significant differencewere found between the'ipsilateral C7'and'oberlin'groups (Z-value were 2.343,0.613,1.143,1.375 respectively,P＞0.017) .Except that the thruput of regeneratingmedullated musculocutaneous nerve fibers of'ipsilateral C7'group was superior tothat of'phrenic nerve'group (Z-value were 3.533,P＜0.017) ,neurophysiological,histological and functional outcome of the musculocutaneous nerve and bicepsshowed no significant difference were found between the'ipsilateral C7'group andother three groups (P＞0.017 ) .6 weeks after operation,Terzis grooming outcome showed the'ipsilateralC7'group was superior to the other three groups (Z-value were 3.252,3.272,3.752respectively,P＜0.017) ,and Ochaii clinical score showed the'ipsilateral C7'groupwas superior to the'forsake repairing axillary nerve'group either.While,the restresults of the behavioral evaluations showed no significant difference were foundbetween the'ipsilateral C7'group and other three groups (P＞0.017)Neurophysiologic and histological investigations of the axillary nerve showed the'ipsilateral C7'group was superior to the other three groups (Z-value were 3.021,3.675,3.782,2.531,3.677,3.780,2.870,3.576,3.674respectively,P＜0.017) .Muscular histological and functional outcome of deltoid muscle showed the'ipsilateral C7'group was superior to the'phrenic nerve'and the'forsake repairingaxillary nerve'groups (Z-value were 3.674,3.780,3.593,3.705,3.285,3.893respectively,P＜0.017) ,but showed no significant difference between the'ipsilateralC7'group and the'oberlin'group (Z-value were 2.286,1.306,1.846 respectively,P＞0.017) .Except that regenerating myelinated axon thruputs of musculocutaneousnerve of the'ipsilateral C7'group were superior to those of three other control groups (Z-value were 3.184,3.674,3.780 respectively,P＜0.017) andneurophysiologicinvestigations of musculocutaneous nerve of the'ipsilateral C7'group were superiorto those of the'forsake repairing axillary nerve'group (Z-value were 3.326,2.797respectively,P＜0.017),rest outcomes of neurophysiologic,histological and functionalinvestigations showed no significant difference were found between the'ipsilateralC7'group and other three groups (P＞0.017 ) .At 12 weeks postoperatively,nearly all the behavioral,neurophysiological,histological and functional determination showed the'ipsilateral C7'group wassuperior to the other three groups (P＜0.017) ,except that no significant differencewere found between the'ipsilateral C7'and other three groups in recovery rate of wetbiceps muscle weight (Z-value were 0.839,1.722,2.340 respectively,P＞0.017),between the'ipsilateral C7'group and the'oberlin'group in recovery rate of latentperiods of CMAP with axillary nerve ,cross-sectional area of biceps muscle fibers andmaximum titanic contractile tension of biceps (Z-value were 1.752,1.810,1.547respectively,P＞0.017) ,between the'ipsilateral C7'group and two control groupsincluding the'oberlin'and'phrenic nerve'groups in recovery rate of CMAP withmusculocutaneou nerve (Z-value were 1.987,2.341 respectively,P＞0.017) andbetween the'ipsilateral C7'group and two control groups including the'phrenicnerve'and'forsake repairing axillary nerve'groups in recovery rate of amplitudes ofCMAP with musculocutaneous nerve (Z-value were 2.135,2.188 respectively,P＞0.017)Conclusions Ipsilateral C7 transferred to the upper trunk of brachial plexuscombined with the spinal accessory nerve to the suprascapular nerve is found to besignificantly effective on treatment of the root avulsion of C5 and C6 of the brachialplexus,which has an better therapeutic effect than other three commonly used nervetransfers. Part 2 An experimental study on mechanism of functionalcompensation for the injured extremity with brachial plexus uppertrunk avulsion after ipsilateral C7 transfer.Objective It was well documented by the follow-up materials that ipsilateralC7 transection had not apparent impairment on the injured-extremity in patients withroot avulsion of C5-C6 of the brachial plexus.The following study was to investigatethe compensative mechanism of no further impairment of the upper limb afteripsilateral C7 transfer for treatment of root avulsion of C5 and C6 of the brachialplexus,which could provide theoretic evidence of generalization for the ipsilateral C7root transfer to treat avulsion of the brachial upper trunk.Methods 60 SD rats were divided into 2 groups,one undergoing transection ofipsilateral C7 root after C5-C6 root avulsion as experimental group,and the othersimple C5-C6 root avulsion as control group.All the rats were fastened supine andanesthetized,then a midline cervical incision was made.After pectoralis major wasincised along the arcuate line,pectoralis minor was retracted laterally,and C5-C7nerve roots were exposed between anterior and middle scalenus muscles.After C5 andC6 nerve roots were avulsed,the ipsilateral C7 root was truncated by one-3mm-section(blocked by 1% procaine),and the neural stumps were sutured to preventthem to regenerate.Neurotization outcomes of the 2 groups were evaluated atpost-operative 3,6 and 12 weeks by comparing the electrophysiologic,histologic,functional and histomorphometric changes of C7-innervated nerves includingthoracodorsal nerve and radial nerve and C7-innervated muscles including latissimusdorsi,triceps brachii ,extensor carpi radialis brevis and extensor digiti communismuscles.The parameters were listed as follows:(1) CMAP:Maximal current andsingle square-wave were used to stimulate the thoracodorsal nerve and radial nerve.The needle electrodes were inserted 2 mm-depth for the four representive muscles,thestimulating and recording electrodes were placed in the same position at bothforelimbs of rats,and the CAMP amplitude and latency were recorded.(2) Musclefunction,regenerating myelinated axon counts,wet weight and cross-sectional area ofmuscle fiber.The distal portions of four representive muscles were free,transectedand attached to the force transducter.The thoracodorsal and radial nerves were simulated respectively with a super-high voltage and a high frequency series of squarewave impulses.The curves of maximum titanic contractile tension of isometriccontraction were recorded on a two-conduction physiological recorder.And then ,thoracodorsal and radial nerve specimens were taken around the anastomotic site.Sections of each nerve were stained with toluidine blue and processed forcomputer-aided image analysis system of regenerative axon number.At last,the fourrepresentive muscles were harvested completely by removing connective tissues,thenweighed by Analytic Balance.Transverse sections of those muscle samples werestained by hematoxylin and eosin (HE),and image analysis systemwas used tomeasure the cross-sectional area of muscle fibers.(3) Synapses distribution and postsynaptic membrane area of muscles.Nerve tissue of entry point to the muscle wereselected to make longitudinal frozen sections.α-bungarotoxin(α-BTX) were incubatedand measured by immunofluorescence staining.The motor end plates distributionfeatures were observed under fluorescent microscope and the overall areas of themwere calculated by imaging analysis system.The average area was obtained by overallareas divided by the number of the post synaptic membrane.(4) Ultramicrostructureof the nerves and muscles.Nerve tissue of entry point to the muscle was dehydratedby ethanol and propanone.After embedding and solidification,ultrathin sections weremade and double-dyed by uranyl acetate-lead citrate.The Morphology of skeletalmuscle motor end plates,i.e.the structure of the myofilament anterior synapticmembrane,the post synaptic membrane and the sarcomere,was observed undertransmission electron microscope.The left forelimbs of both group rats were taken asthe experimental sides,and the right forelimbs of them were taken as the control sides.Values of the control side were taken as 1,so that the recovery rates of the left sidecould be obtained by the parameters of the left forelimb divided by those of the rightforelimb.Those recovery rates between the C7-transection and control groups werecompared by Wilcoxon signed rank test.P-value＜0.05 were considered as significantlevel.Results 3 weeks after operation,recovery rates of amplitude of CMAP andCMAP latency of those four representive muscles in the C7-transection group weresignificantly lower than those in the control group(p＜0.05).Recovery rates ofmuscular wet weight,muscular contractile tension and cross-sectional area of musclefibers of four representive muscles in the C7-transection group were also significantlylower than those in the control group(p＜0.05).In addition,the recovery rate of area of post synaptic membranes of those four muscles in the C7-transection group wassignificantly lower than that in the control group(p＜0.05).The abnormalultramicrostructure of the motor end plates of four representive muscles was found inthe C7-transection group,that is,immature morphology,smaller but integralmicrostructure in the anterior synaptic membrane area,less folds and absence of deepgrooves in the post synaptic membrane,narrower synaptic cleft,irregularly orderedmyocomma and myofilament.Motor end plates in part of latissimus dorsi and oftriceps were still in a primary stage of morphology in the C7-transection group.At 6 weeks postoperatively,recovery rates of CMAP amplitude and latency fortriceps were not significantly different between the C7-transection and controlgroups(Z-value were 1.143,1.633 respectively,p＞0.05).For extensor carpi radialisbrevis and extensor digitorum communis,recovery rates of cross-sectional area ofmuscle fibers,of the amplitude and latency of CMAP and of the area of post synapticmembranes were not significantly different between the two groups (Z-value were1.715,2.206,0.735,1.061,2.124,2.613,2.165,2.776 respectively,p＞0.05),while,the rest of parameters of evaluation were still significantly different between the twogroups(p＜0.05).As far as the ultramicrostructure was concerned in the C7-transectiongroup,more motor end plates of four representive muscles were observed,and theirultramicrostructure also had a tendency to mature as compared with those at 3 weekspostoperatively.12 weeks after operation,all parameters of the C7-transection group were notsignificantly different with those of the control group(p＞0.05).Motor end plates weredensely distributed and their ultramicrostructure in four representive muscles ofC7-transection group appeared to be mature as compared with those of the controlgroup,i.e.large area of anterior synaptic membrane containing chondriosomes,microtubules and abundant synaptic vesicle,deep folds in post synaptic membrane,clear synaptic cleft,well-ordered myocomma and myofilament without swelling ordisruption.Conclusions After ipsilateral C7 transfer for treatment of root avulsion ofC5-C6 of the brachial plexus,the nerve fibers of lower trunk can compensativelyinnervate fibres of C7-representive muscles by means of neuraxon gemmation andmotor end plate regeneration,so there is no further impairment in the affectedextremity.